Gene therapy against HIV infection holds the promise of providing alternatives to traditional drug based therapies. Ultimately, such therapy may be able to prevent progressive HIV infection by sustained interference with viral replication in the absence of chronic chemotherapy. The experiments described in this proposal are aimed at providing a detailed understanding of the mechanism of action of an HIV antisense construct that efficiently inhibits HIV replication. In preliminary studies, we have obtained evidence to suggest that efficient antisense inhibition is explained by (and dependent on) trafficking of the antisense RNA through the Rev/RRE pathway. Significantly, these studies also suggest a novel cytoplasmic mechanism for antisense inhibition. The application has three specific aims which are to: 1) Further analyze the target Ribonuclear Protein (RNP) complexes that are present in cells expressing RRE-driven antisense RNA;2) Investigate how RRE-driven antisense inhibition relates to cellular small regulatory RNA pathways;3) Determine if the ADAR editing machinery is involved in antisense inhibition. These studies are of importance for further development of therapeutic vectors delivering antisense RNA, since they may lead to the conclusion that there is a significant advantage in making sure that antisense RNA for HIV therapy traffics along the Rev/RRE pathway. In addition, these studies will undoubtedly give us further insight into the interactions between Rev and the cellular machinery. If we can establish a connection between Rev/RRE, the ADAR editing machinery and the generation of miRNA, this would have even more significant implications. Such findings would provide the basis for further studies looking into the possibility that HIV uses Rev and the RRE to increase viral diversity through ADAR editing. PUBLIC HEALTH RELEVANCE: This research will focus on a novel therapeutic approach for HIV infection, which involves the use of antisense RNA. We will investigate the mechanisms underlying the efficient inhibition of HIV that is seen when this RNA is expressed in infected cells and the relationship of these mechanisms to the HIV Rev-RRE mediated nucleo-cytoplasmic transport pathway. The work has significance for the development of novel HIV therapeutic and also for the general development of the use of antisense strategies for treatment other diseases.